ABSTRACT Light detection in rod photoreceptors is mediated by a G protein-signaling cascade triggered when the visual pigment rhodopsin, a prototypical G protein-coupled receptor, is activated by light and initiates the rod light response. Eventually, the photoactivated rhodopsin decays to all-trans-retinal and free opsin. The chromophore-free opsin produces persistent transduction activity even in darkness. This, in turn desensitizes the photoreceptors and limits our ability to see following exposure to bright light. Abnormally high opsin activity due to opsin mutations or slow pigment regeneration can be detrimental to rod function and survival. Despite the role of opsin activity in modulating rod physiology in normal and disease conditions, the molecular mechanism by which opsin stimulates rod transduction has remained unclear. The prevailing view is that each opsin has low uniform constitutive activity. We will evaluate an alternative hypothesis that, similar to rhodopsin, opsin exists in equilibrium between a distinct inactive state and a rare but highly efficient active state. This hypothesis is based on our preliminary data showing that introduction of a small amount of free opsin by rhodopsin bleaching results in the generation of discrete, photoresponse-like events in mouse rods. We will perform experiments to determine the amplitude and kinetics of the quantal response produced by a single opsin molecule in mouse, primate, and human rod photoreceptors. We will also determine the mechanism of modulation of opsin activity by non-covalent binding of chromophore analogs and chaperones, and whether blocking opsin signaling either by quenching with chaperones, or by using 5- or 6-locked rhodopsin that does not dissociate upon photoactivation restores the sensitivity of chromophore-deficient rods. Finally, we will perform experiments to determine the role of phosphorylation and arrestin binding in the inactivation of opsin signaling. These experiments will establish the molecular mechanisms by which opsin activates the rod transduction cascade to produce bleaching adaptation. They will also help us understand how this activity can be modulated pharmacologically, potentially leading to the development of treatments for a range of opsin-related visual disorders such as congenital stationary night blindness and retinitis pigmentosa.